Draft genome sequence of Enterobacter cloacae ST473 harbouring blaCMH-3 isolated from a human patient diagnosed with recurrent bacteriuria in Nigeria

Enterobacter cloacae is among the most frequently isolated species described in clinical infections and is commonly associated with a multidrug resistance (MDR) phenotype. We present the draft genome sequence of a MDR E. cloacae isolated in Nigeria from the urine sample of an adult male outpatient diagnosed with symptomatic recurrent bacteriuria. The isolate was found to be resistant to ceftriaxone, cefotaxime, cefepime and levofloxacin. Genome analysis revealed the presence of the beta-lactamase chromosomal gene blaCMH-3, which may be responsible for the antibiotic resistance observed in the recurrent E. cloacae urinary tract infection.

despite the detection of this blaAmpC variant in several publicly available E. cloacae genomes [2]. Here we report the draft genome sequence of an E. cloacae isolate collected in March 2021 at the Laboratory unit of Alex Ekwueme Federal Teaching Hospital Abakaliki, Ebonyi State, Nigeria from the urine sample of an adult male outpatient with recurrent urinary tract infection (UTI).
The isolate was collected, stored in nutrient broth with glycerol. Antibiotic susceptibility testing was performed by the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines 2021 on Muller-Hinton (MH) agar [5]. After 24 h of growth at 37 °C, the isolate was evaluated for its susceptibility to ciprofloxacin (5 µg), levofloxacin (5 µg), cefepime (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), amoxicillin-clavulanic acid (20/10 µg), ampicillin (10 µg), imipenem (10 µg) and fosfomycin (200 µg). A modified DDST (modified double disc synergy test) with the use of cefepime was also performed to improve the detection of ESBL-producing isolates, which co-produce AmpC [6] (Fig. S1, available in the online version of this article). The isolate was found to be resistant to the two third generation cephalosporins (ceftriaxone and cefotaxime) and showed intermediate resistance (MIC of 15 µg ml −1 with zone of inhibition of 18.4 mm ±0.01) to cefepime, a fourth-generation cephalosporin. The isolate was susceptible to amoxicillin-clavulanic acid and imipenem, and resistant to ampicillin. Regarding the fluoroquinolones, the isolate showed intermediate resistance to ciprofloxacin (MIC of 0.5 µg ml −1 with zone of inhibition of 23 mm ±0.00) and resistance to levofloxacin. We also verified that the isolate showed resistance to fosfomycin.
The isolate was cultured in Tryptic Soy Agar (TSB) broth and 0.5 ml was used for genomic DNA extraction with the Wizard Genomic Purification kit (Promega, USA). Purified DNA was used as a template for 16S rRNA gene amplification with universal primers 27F (5ʹ AGAGTTTGATCMTGGCTCAG-3ʹ) and 1525R (5'-AAGGAGGTGWTCCARCCGCA-3′) as previously described [7]. The amplicon was subjected to sequencing reactions using Big Dye terminator v3.1 cycle sequencing kit (ThermoFisher Scientific) with 27F or 1525R as sequencing primers and then injected on an ABI PRISM 3130XL genetic analyser (ThermoFisher Scientific). The obtained sequences were used to assemble a 16S RNA 1546 bp sequence, which was then blastn searched against the GenBank/NCBI database allowing taxonomic identification of the clinical isolate as E. cloacae (100 % identity, 100 % query cover).
Whole-genome sequencing of the E. cloacae isolate was performed on Illumina MiSeq platform (Illumina, CA, USA) at the Center for Advanced Technologies in Genomics (CATG), Institute of Chemistry, University of Sao Paulo, using the genomic DNA purified with Wizard Genomic Purification kit (Promega, USA) DNA. The shotgun genomic library was prepared using the Nextera DNA Library Prep (Illumina) with a total DNA input of ~20 ng and subjected to a run using an Illumina MiSeq Reagent Kit v3 (2×300 cycles). The reads were checked for quality phred=30 and filtered using Fastp v0.12.4 [8]. Filtered rawR1 reads were used for de novo assembly using SPAdes v3.15.4 [9]. Assembly completeness and contamination were verified with CheckM v. v1.2.0 [10] and the assembly quality was evaluated with quast v5.0.2 [11]. The assembled genome was annotated using prokka v.1.14.6 [12]. General features of the genome assembly and annotation are presented in Table 1.
Sequence type was defined using MLST v 2.0.9 and multi locus sequence typing (MLST) allele sequences and profile data obtained from PubMLST. org [13,14]. ResFinderFG v 2.0 was used to identify antibiotic resistance determinants [15]. MobileElementFinder v 1.0.3 and ISfinder were used to identify insertion sequences and mobile genetic elements and their relation to antimicrobial resistance genes and virulence factors [16,17]. Table 2 lists the antibiotics resistance genes (ARGs) detected in the E. cloacae draft genome. It is worth noting a chromosomal blaCMH-3 gene with 100 % identity and 100 % coverage to the reference gene. Hence, this is the first time multidrug-resistant E. cloacae with an ESBL phenotype harbouring a single narrow spectrum blaCMH-3 betalactamase is reported in Nigeria. A fosfomycin resistance gene fosA was also detected. Genes of the RND (resistance nodulation division) drug efflux system, OqxAB, related to fluoroquinolone resistance was also observed. The E. cloacae isolate seems to carry a plasmid without antibiotic resistance genes, which is similar to plasmid ColRNAI originally found in Klebsiella pneumoniae strains where it encodes carbapenems resistance genes [18]. The genomic features of E. cloacae clinical isolate described here suggest that a single AmpC beta-lactamase may be responsible for a broader extended spectrum beta-lactamase drug resistance. Despite the cost associated to whole-genome sequencing of clinical isolates, which might be prohibitive in regions with limited resources, elucidating the genetic basis of resistance is highly valuable to enable initial antibiotic selection to minimize use of ineffective antibiotics [19]. Based solely on the susceptibility testing phenotypic assays, a successful treatment of the recurrent UTI reported in this study could be achieved with amoxicillin-clavulanic acid. However, in vitro susceptibility does not necessarily translate to in vivo susceptibility. This may be related to the current clinical practice of high amoxicillin-to-clavulanate ratios, which has been found to result in the most rapid adaptation to antibiotics via gene dosing responses [20]. Comments: The authors have effectively addressed the feedback provided during the initial review. Given that this article presents a concise communication announcing the genome of a clinically significant bacterium, I highly recommend its publication in the journal. However, I do suggest further refinement of the language to enhance the clarity of the message through a thorough language review.

Please rate the manuscript for methodological rigour Good
Please rate the quality of the presentation and structure of the manuscript Satisfactory To what extent are the conclusions supported by the data? Strongly support Thank you for your email of Apr 14, 2023. We thank you and the Reviewers for the time taken to read our manuscript and for the insightful comments and valuable suggestions that were provided which were helpful for improving the manuscript.

If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines? Yes
We believe we have responded and clarified all the points raised by the reviewers. The changes we have made are highlighted in blue in the manuscript file as well as duly addressed in our point-by-point responses to the reviewers. As a result, this version of the manuscript is an improvement with respect to the previous one.
We hope the changes and corrections we have made are satisfactory. We look forward to hearing from you as to the suitability of our manuscript for publication.

Reviewer 2
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines? Reviewer 2 Comments to Author: The article is a genome announcement. The use of genome analysis to identify the resistance mechanism is a commendable approach, as it provides a detailed understanding of the genetic basis of resistance.The presence of the beta-lactamase chromosomal gene blaCMH-3 is an important finding, as it sheds light on the mechanism of resistance to beta-lactam antibiotics in this isolate.
The findings of this study could have implications for the management of patients with recurrent urinary tract infections caused by MDR E. cloacae, particularly in regions with limited resources for microbiological testing and antibiotic susceptibility testing.
The article does not discuss any potential limitations of the study, such as selection bias, potential contamination of the sample, or limitations of the genome analysis.Such discussion would is of importance to evaluate the robustness of the findings and their implications for clinical practice.

R:
We thank the reviewer for the positive evaluation of our work, particularly the contribution of this study for the management of patients with recurrent urinary tract infections caused by MDR E. cloacaein regions with limited resources for microbiological testing and antibiotic susceptibility testing.As suggested,we have added a brief discussion about potential limitations of the study (please see lines 119-127).

Further I have noted some typographical errors
Eg;-Enterobacter cloacae is written in different ways eg : 'cloacae' in line 31 and 'cloaca' in line 58, please strict to one correct way.

R:The typographicalerror has been corrected (please see line 59). The text was further checked for typos.
In summary, the article comprised of a concise genome announcement which is of a an organism with some clinical implications. I would suggest to expand the article including the potential limitations and to correct the grammar and spelling errors.
R:As mentioned above we have added a brief discussion about potential limitations of the study (please see lines 119-127). The text was further checked for grammar and spelling errors.